The overall objective of this proposal is to understand the structural basis for the divergent functions of the kinesin family of motor proteins. Kinesin-family motor proteins are defined by an evolutionary conserved motor domain that uses the energy of ATP hydrolysis to power organelle transport, cell motility, and mitosis, through interactions with the microtubule cytoskeleton. To perform these functions, the family includes proteins with very different activities. Conventional kinesin walks along the microtubule lattice, while MCAK, a member of the kinesin-13 family of proteins, diffuses on the microtubule lattice and depolymerizes microtubules from both ends. The yeast protein Kip3 (kinesin-8) does both; it walks to the plus-end of the microtubule and then removes tubulin dimers. Which structural features of these motor proteins explain their different behaviors? I will address this question using a single molecule in vitro assay based on total-internal- reflection-fluorescence (TIRF) microscopy. The assay will be performed on native kinesin, MCAK, and Kip3 as a positive control. Then, using mutant proteins with modified structural features, I will test (a) the electrostatic tether model for the diffusive MCAK/microtubule interaction and (b) the hypothesis that unique features in the motor domains of kinesin-13 and kinesin-8 confer depolymerase activity. The results will identify the structures responsible for translocase activity, lattice diffusion, and depolymerase activity. [unreadable] [unreadable] [unreadable]